Donald R. A. UgesLaboratory for Drug Analysis and Toxicology, Department of Pharmacy, University Hospital Groningen and School of Pharmacy University of Groningen, Groningen, The Netherlands

A simple definition of intoxication might be: "Just too much of an exogenous substance." But what is too much?

A patient suffering from severe pain from cancer, gets more and more morphine. Then he dies. Is this death caused by intoxication, or by a required treatment with lethal side effects?

Somebody puts a sleeping pill in a glass of whisky. After drinking this glass this victim fell asleep and was raped. Is this real intoxication, considering that sleeping is the desired effect of a sleeping pill?

A German driver has a legal level of 0.7 g/l ethanol in her blood. She crosses the border to Holland and causes an accident. (Driving in Holland with a blood alcohol level of 0.7 g/l is illegal). Is the woman intoxicated?

A man smokes twenty cigarettes a day without any clinical symptoms. Is he intoxicated?

An aggressive junkie with a high, but not lethal, cocaine blood level is arrested by several policemen. Then he suddenly dies due to the combination of cocaine and stress. Who is responsible for this death?

In view of prevailing knowledge and today's medical practice I would like to propose the following definition of 'intoxication': 'A medically or socially unacceptable state, into which an individual has fallen, caused by too high a dose of an exogenous substance taken by the person him/herself or given by another person.'

Keywords: Intoxication, Definition.

An Attempt to Improve the Interpretation of
Post-Mortem Drug Levels by the Creation of a National
Drug-Related Death Database

The lack of complete data from death by poisoning (drug or other material) is one factor affecting the confidence that the toxicologist can attach to the interpretation of post-mortem drug levels. The UK Forensic Toxicology Forum is a group of major forensic and clinical laboratories within the United Kingdom. One of the projects of this group is an attempt to create a database of drug-related deaths that will enable dynamic selection of data to create probabilities and confidence levels specific to particular cases.

Drug-death data and toxicological categories (therapeutic, toxic, and fatal) are often presented as ranges. The biological reality is more likely to be a series of normal distributions for each category. Our database seeks to establish these distributions and allow recording and inclusion of other case related information such as time between death and analysis. This approach allows the toxicologist to interrogate the database for cases similar to the case under investigation.

For each drug, and each drug concentration, the database will yield a probability that this is within the therapeutic, toxic, or fatal range. It will also yield a confidence determined by the number of cases that the toxicologist's search criteria have found. The stringency of the search may be reduced to increase the confidence.

It would be valuable to present this in the scientific session to seek international partnerships in this important area or to identify and learn from similar projects elsewhere.

Keywords: Drug Death Database.

A Two-Fold Variation in the Ratio between the Active and Inactive Enantiomers of the Antidepressant Citalopram in Forensic Autopsy Cases

Citalopram is a selective serotonine reuptake inhibitor used in patients with depressive disorders and panic syndrome. Citalopram is fairly frequently found in the screening for pharmaceutical drugs undertaken in more than 90% of the forensic autopsies in Sweden. Citalopram is a racemate and the therapeutic effect resides in the S-(+)-citalopram. The compound is metabolized to desmethylcitalopram and further to didesmethylcitalopram, both of which are racemates.

Enantioselective analysis of citalopram is used to study the variation in the ratio between the active S-(+)-citalopram and the inactive enatiomer with special emphasis on a possible correlation between the racemate concentration and the enantiomer ratio.

Citalopram racemate concentrations ranged from 0.5 µg/g blood to 38.9 µg/g blood. The ratio between S- and R-citalopram showed a 2-fold variation, from 0.54 to 1.06 (mean 0.8, SD 0.18): The ratio between S-citalopram and racemic citalopram ranged from 35 to 52%, mean 45%. This is higher than has been reported in patients at steady-state. No correlation was found between the enantiomer ratio and the racemate concentration. The concentration of desmethylcitalopram ranged from 0.08 to 2.92 µg/g blood (as the racemate). The ratio between S- and R-desmethylcitalopram ranged from 0.44 to 1.07. The concentration of didesmethylcitalopram was under the limit of detection (0.002 µg/g blood) in most cases. The ratio between racemic citalopram and racemic desmethylcitalopram varied between 0.9 and 20! In patients at steady-state, ratios between 2 and 4 are common.

There is a 2-fold variation in the ratio between S- and R-citalopram in the cases studied. This is in agreement with data from clinical studies. In individual cases it might be of value to perform enantioslective analysis. Citalopram is metabolized via CYP2D6 and CYP2C19 and it ought to be studied if poor metabolizers of either enzyme show different ratios between the enantiomers.

Whatever its THC concentration, hemp can be legally cultivated in Switzerland provided it is not used for narcotic production. Five g of a commercially available hemp tea containing 35 mg total THC (0.7%) were infused either in 2 dl of hot water or milk for a half an hour, filtered and administered to 2 groups of 6 healthy volunteers. Half of them drank the water infusion while the 3 others ingested the milk hemp tea. Two weeks later, the experiment was repeated with the same volunteers, but this time the type of hemp decoction which was administered was changed. Before administration and during the whole study, biological samples were drawn periodically. Physiological measures included heart rate and conjunctival injection. Subjective effects (rating "high" and sedation) were assessed with a Visual Analog Scale (VAS). Cannabinoids were determined by immunoassays (RIA, EIA) and GC/MS after solid-phase extraction. The presence of cannabinoids in the sweat was tested by immunoassay (Drugwipe®).

Because of its lipophilic character, much higher levels of THC were extracted from hemp into milk (21 mg THC) compared to water (1.7 mg) during the tea-making process. Peak levels of cannabinoids determined in urine by RIA following ingestion of the hemp milk decoction (1400-10,700 ng/ml) were much higher than those measured after drinking the water infusion (104-265 ng/ml). A similar picture was obtained when carboxy-THC was quantified by GC/MS: maximum carboxy-THC concentrations were in the range of 346-1586 ng/ml following milk tea intake and between 16 and and 85 ng/ml urine after drinking the water infusion. GC-MS determinations of THC, 11-OH-THC and carboxy-THC revealed the presence in the whole blood of only very low levels of carboxy-THC (less than 5 ng/ml) after drinking the water
infusion while significant concentrations of all these 3 cannabinoids were determined after intake of the milk infusion. The highest mean concentrations of THC, 11-OH-THC and carboxy-THC were 4.0, 3.4 and 24.5 ng/ml, respectively. Trace amounts of cannabinoids could only be found in the saliva collected a short while after ingestion. In contrast to blood, urine and saliva, no cannabinoids could be detected in the sweat collected on the forehead of the volunteers. Blood levels measured at different time-intervals were used to calculate the time of hemp tea administration with the mathematical models
developed by Huestis & al. The quality of fit of the models to the data will be discussed.

In order to correctly interpret forensic toxicological results it is important to know the stability of certain compounds. Normally there is a delay of some days between sampling and analysis and in some cases a new question may arise whether a drug was present or not several weeks or months later. The aim of the present study was to evaluate the consequence of long-lasting storage of blood samples with special attention to the stability of various drugs.

Material and methods: An autopsy femoral blood and vitreous humor were collected and forwarded to the laboratory of forensic chemistry. The vitreous humor was divided into two portions and potassium fluoride was added as a preservative to one of the samples as well as to the femoral blood samples. Analysis for drugs in the femoral blood was made within a week or less from the arrival of the sample. The vitreous humor was kept at +4°C for about a month until stored at -20°C. After analysis the blood samples were stored at -20°C. For most drugs, analyses were performed by gas-chromatography and nitrogen sensitive detector. The first analyses were normally performed within two days after the arrival of the sample and the sample was reanalyzed one year later using the same analytical method. The samples from the vitreous humor were only analyzed after one year of storage.

Results: In the present study, we have examined the stability of different kind of drugs including benzodiazepines; analgesics like propoxyphene and acetaminophen; antidepressants like citalopram and amitriptyline; antiepileptics; and hypnotics. In femoral blood, no major changes were seen for most of the drugs studied except for 7-amino-nitrazepam where a significant loss was detected after one year of storage. In the vitreous humor no major differences were noted between the samples with and without preservative. This implies that addition of preservative to vitreous samples does not seem to be necessary. The use of preservative-free samples will also allow analysis for electrolytes e.g. potassium and sodium, which is sometimes important. Generally, the concentration was lower in vitreous compared to femoral blood. However, the concentrations of citalopram and its metabolite desmethylcitalopram were fairly equal in vitreous and the blood.

Conclusions: Storage of blood samples, with a preservative like potassium fluoride added, for one year at -20°C does not affect the concentration except for some drugs like nitro-benzodiazepines. Knowledge of the stability of drugs in stored samples is of great importance when interpreting toxicological results. Vitreous humor could be an alternative medium for performing toxicological analyses, provided the ratio of the drug concentration in the blood to that in vitreous humor
is known.

Statistical Estimation of High Alcohol Concentrations
Determined in Breath and in Blood

In the study an attempt was made to compare the blood alcohol concentrations obtained from the analysis of breath and from direct blood investigation.

Blood samples were taken from those patients of the Sobering Chamber in Cracow in whom blood alcohol concentration determined by the analysis of breath exceeded 3o/oo. 56 results of simultaneous determinations of alcohol in blood samples and in breath were taken for the comparison.

Breath alcohol concentrations were determined using Alcomat V5, which utilises selective absorption in the IR region. Blood alcohol investigations were carried out by means of gas chromatography with the use of headspace technique.

The results of breath-alcohol analysis differed from -0.83o/oo to 1.37o/oo in comparison with the results of direct blood investigation. The relative difference between these two variables ranged from 18.6% to 54.8% for particular patients.

The correlation between the results of breath- and blood-alcohol analyses were evaluated. The correlation coefficient amounted to 0.242 and was statistically insignificant (p>0.05). Furthermore, the slope and the intercept of the straight line adjusted to the experimental data were significantly different from the expected values (tb0=11.35, tb1=11.16, whereas t0.05,54=1.98). This might indicate a very low precision of the results of breath analyses at high alcohol concentrations.

Moreover, the blood/breath ratios were calculated for each patient. The mean value of this coefficient for the investigated group amounted to 2063:1 +/- 230 (1 standard deviation), and the coefficient of variation amounted to 11.2%. Although the mean value of the blood/breath ratio is nearly the same as the one used in Alcomat, it varies depending on the patient from 1357:1 to 2580:1.

The results of the study show that there is no justification for continuation of the current practice which consists in treating alcohol blood concentrations obtained by the analysis of breath as equivalent to alcohol concentrations determined in the direct analysis of blood samples.

Keywords: Ethanol, Breath Analysis, Blood, Correlation.

The Accuracy of Sequences of Steps in Analytical Procedures in Forensic Toxicology. Its Significance in the Interpretation of the Final Results of Individual Cases of Poisoning

The level of accuracy/uncertainty of the determination of a xenobiotic in postmortem tissue will depend on the least precise stage of the analytical procedure. This commonly known factor affects the final results of an analysis in individual cases of poisoning, and hence their interpretation.

In the sequence of analytical operations the final steps of the toxicological analysis of purified and homogenous samples are highly precise. Modern physico-chemical methods allow a high degree of repeatability. The step of isolating the substance is characterised by a much wider variability. In certain cases estimates may be made. But in the process of preparing expert opinions, the significance of this variability is often overlooked. The reasons might include the time-consuming nature and high cost of an accurate determination of repeatability in every single case at this stage of analysis. The biological variability encompasses a wide range and blurred boundary values of concentration. This has made it difficult to categorise the result to a precise interval, which will affect its interpretative evaluation.

This will be made even worse by an inaccurate definition of samples at the initial stage and by the credibility of the samples - associated with the distribution of the xenobiotic within the organ. This issue is not highlighted in the opinions provided by forensic experts. This is probably caused by an intuitive feeling of the inexpedience of trying to make the process of isolation highly precise, with the well-justified assumption that it will disappear later. This inconsistency leads to unreliability in the accuracy of the final result, which does not reflect the total uncertainty.

One should emphasise the need for a holistic approach to the process of forensic toxicological analysis and for attributing various levels of variability to its subsequent stages. The fierce competition in the field of the most modern methods is not justified without paying attention to the whole process. One issue should be particularly highlighted: at which stages will improvement in variability depend on the expertise of the expert, and at which stages is the level of our knowledge the limiting factor?